DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : III. VISCOSITY.

1. Gelatin solutions have a high viscosity which in the case of freshly prepared solutions varies under the influence of the hydrogen ion concentration in a similar way as the swelling, the osmotic pressure, and the electromotive forces. Solutions of crystalline egg albumin have under the same conditions a comparatively low viscosity which is practically independent of the pH (above 1.0). This difference in the viscosities of solutions of the two proteins seems to be connected with the fact that solutions of gelatin have a tendency to set to a Jelly while solutions of crystalline egg albumin show no such tendency at low temperature and pH above 1.0. 2. The formulæ for viscosity demand that the difference in the order of magnitude of the viscosity of the two proteins should correspond to a difference in the relative volume occupied by equal masses of the two proteins in the same volume of solution. It is generally assumed that these variations of volume of dissolved proteins are due to the hydration of the isolated protein ions, but if this view were correct the influence of pH on viscosity should be the same in the case of solutions of gelatin, of amino-acids, and of crystalline egg albumin, which, however, is not true. 3. Suspensions of powdered gelatin in water were prepared and it was found, first, that the viscosity of these suspensions is a little higher than that of gelatin solutions of the same concentration, second, that the pH influences the viscosity of these suspensions similarly as the viscosity of freshly prepared gelatin solutions, and third, that the volume occupied by the gelatin in the suspension varies similarly as the viscosity which agrees with the theories of viscosity. It is shown that this influence of the pH on the volume occupied by the gelatin granules in suspension is due to the existence of a Donnan equilibrium between the granules and the surrounding solution.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : IV. VISCOSITY—Continued.

1. The proof is completed that the influence of electrolytes on the viscosity of suspensions of powdered particles of gelatin in water is similar to the influence of electrolytes on the viscosity of solutions of gelatin in water. 2. It has been suggested that the high viscosity of proteins is due to the existence of a different type of viscosity from that existing in crystalloids. It is shown that such an assumption is unnecessary and that the high viscosity of solutions of isoelectric gelatin can be accounted for quantitatively on the assumption that the relative volume of the gelatin in solution is comparatively high. 3. Since isoelectric gelatin is not ionized, the large volume cannot be due to a hydration of gelatin ions. It is suggested that this high volume of gelatin solutions is caused by the existence in the gelatin solution of submicroscopic pieces of solid gelatin occluding water, the relative quantity of which is regulated by the Donnan equilibrium. This would also explain why the influence of electrolytes on the viscosity of gelatin solutions is similar to the influence of electrolytes on the viscosity of suspensions of particles of gelatin. 4. This idea is supported by experiments on solutions and suspensions of casein chloride in which it is shown that their viscosity is chiefly due to the swelling of solid particles of casein, occluding quantities of water regulated by the Donnan equilibrium; and that the breaking up of these solid particles into smaller particles, no longer capable of swelling, diminishes the viscosity. 5. This leads to the idea that proteins form true solutions in water which in certain cases, however, contain, side by side with isolated ions and molecules, submicroscopic solid particles capable of occluding water whereby the relative volume and the viscosity of the solution is considerably increased. This accounts not only for the high order of magnitude of the viscosity of such protein solutions but also for the fact that the viscosity is influenced by electrolytes in a similar way as is the swelling of protein particles. 6. We therefore reach the conclusion that there are two sources for the viscosity of protein solutions; one due to the isolated protein ions and molecules, and the other to the submicroscopic solid particles contained in the solution. The viscosity due to the isolated molecules and ions of proteins we will call the general viscosity since it is of a similar low order of magnitude as that of crystalloids in solution; while the high viscosity due to the submicroscopic solid protein particles capable of occluding water and of swelling we will call the special viscosity of protein solutions. Under ordinary conditions of hydrogen ion concentration and temperature (and in not too high a concentration of the protein in solution) the general viscosity due to isolated ions and molecules prevails in solutions of crystalline egg albumin and in solutions of metal caseinates (where the metal is monovalent) while under the same conditions the second type of viscosity prevails in solutions of gelatin and in solutions of acid-salts of casein; and also in solutions of crystalline egg albumin at a pH below 1.0 and at higher temperatures. The special viscosity is higher in solutions of gelatin than of casein salts for the probable reason that the amount of water occluded by the submicroscopic solid gel particles in a gelatin solution is, as a rule, considerably higher than that occluded by the corresponding particles of casein.     

AN EMPIRICAL FORMULA FOR THE RELATION BETWEEN VISCOSITY OF SOLUTION AND VOLUME OF SOLUTE

It has been found that the expression See PDF for Equation represents very closely the relation between the volume of the solute and the viscosity of the solution. The formula has been applied to a number of experimental results and found to hold very well for as high concentrations as 50 per cent solutions of such substances as sugars, glycogen, casein, and rubber. In the case of various sugar solutions, and also in the case of sulfur suspensions, the volume of the solute as calculated from the viscosity values agrees with the actual volume of the substance in dry state, as determined from specific gravity measurement, while in the case of caoutchouc solutions in benzene the values of ϕ as calculated from the viscosity measurements fit remarkably well in the equation for osmotic pressure.     

Volumetric effects of ionization of amino and carboxyl termini of alpha,omega-aminocarboxylic acids

We have determined the partial molar volumes and adiabatic compressibilities of a homologous series of six alpha,omega-aminocarboxylic acids over a broad pH range at 25 degrees C. We interpret the resulting data in terms of the changes in hydration associated with neutralization of amino and carboxyl termini. By combining our volumetric results with pH-dependent data on 1-anilinonaphthalene-8-sulfonic acid fluorescence we propose the following explanation to the long-standing observation that changes in volume and compressibility accompanying neutralization of a carboxyl group depend on the type of the solute in contrast to solute-independent changes in these parameters accompanying neutralization of an amino group. Unlike amino groups, neutralized carboxyl groups are capable of forming hydrogen-bonded structures stabilized by hydrogen bonds between the carbonyl oxygen of one solute molecule and the hydroxyl group of another molecule. Formation of such hydrogen-bonded structures causes an additional decrease in solute hydration with concomitant increases in volume and compressibility. Furthermore, solutes with large aliphatic moieties may form larger associates stabilized, in addition to intermolecular hydrogen bonds, by hydrophobic interactions which will result in further increases in volume and compressibility. In the aggregate, our results emphasize the need for further studies focused on developing an understanding of the role of electrostatic interactions in stabilizing/destabilizing proteins and protein complexes.     

THE R?LE OF THE ACTIVITY COEFFICIENT OF THE HYDROGEN ION IN THE HYDROLYSIS OF GELATIN

1. The hydrolysis of gelatin at a constant hydrogen ion concentration follows the course of a monomolecular reaction for about one-third of the reaction. 2. If the hydrogen ion concentration is not kept constant the amount of hydrolysis in certain ranges of acidity is proportional to the square root of the time (Schütz''s rule). 3. The velocity of hydrolysis in strongly acid solution (pH less than 2.0) is directly proportional to the hydrogen ion concentration as determined by the hydrogen electrode i.e., the "activity;" it is not proportional to the hydrogen ion concentration as determined by the conductivity ratio. 4. The addition of neutral salts increases the velocity of hydrolysis and the hydrogen ion concentration (as determined by the hydrogen electrode) to approximately the same extent. 5. The velocity in strongly alkaline solutions (pH greater than 10) is directly proportional to the hydroxyl ion concentration. 6. Between pH 2.0 and pH 10.0 the rate of hydrolysis is approximately constant and very much greater than would be calculated from the hydrogen and hydroxyl ion concentration. This may be roughly accounted for by the assumption that the uncombined gelatin hydrolyzes much more rapidly than the gelatin salt.     

DOES THE KINETICS OF TRYPSIN DIGESTION DEPEND ON THE FORMATION OF A COMPOUND BETWEEN ENZYME AND SUBSTRATE

1. The velocity of hydrolysis of gelatin by trypsin increases more slowly than the gelatin concentration and finally becomes nearly independent of the gelatin concentration. The relative velocity of hydrolysis of any two substrate concentrations is independent of the quantity of enzyme used to make the comparison. 2. The rate of hydrolysis is independent of the viscosity of the solution. 3. The percentage retardation of the rate of hydrolysis by inhibiting substances, is independent of the substrate concentration. 4. There is experimental evidence that the enzyme and inhibiting substance are combined to form a widely dissociated compound. 5. If the substrate were also combined with the enzyme, an increase in the substrate concentration should affect the equilibrium between the enzyme and the inhibiting substance. This is not the case. 6. The rate of digestion of a mixture of casein and gelatin is equal to the sum of the rates of hydrolysis of the two substances alone, as it should be if the rate is proportional to the concentration of free enzyme. This contradicts the saturation hypothesis. 7. If the reaction is followed by determining directly the change in the substrate concentration, it is found that this change agrees with the law of mass action; i.e., the rate of digestion is proportional to the substrate concentration.     

A METHOD FOR THE QUANTITATIVE DETERMINATION OF TRYPSIN AND PEPSIN

A quantitative method is described which permits a determination of the relative amount of trypsin or pepsin present in a gelatin-enzyme digestion mixture, provided the gelatin and trypsin solutions are purified. This method is dependent upon the change in viscosity of such solutions. It is found that the time required to cause a given percentage change in the viscosity is nearly inversely proportional to the amount of enzyme present. It is pointed out that the particular value of the method lies in the fact that enzyme reactions which take place in the presence of "buffer" salts may be studied.     

THE SWELLING PRESSURE OF GELATIN AND THE MECHANISM OF SWELLING IN WATER AND NEUTRAL SALT SOLUTIONS

1. A method is described for measuring the swelling pressure of solid gelatin. 2. It was found that this pressure increases rapidly between 15° and 37°C., and that the percentage change is nearly independent of the concentration of gelatin. 3. It is suggested that this pressure is due to the osmotic pressure of a soluble constituent of the gelatin held in the network of insoluble fibers, and that gelatin probably consists of a mixture of at least two substances or groups of substances, one of which is soluble in cold water, does not form a gel, and has a low viscosity and a high osmotic pressure. The second is insoluble in cold water, forms a gel in very low concentration, and swells much less than ordinary gelatin. 4. Two fractions, having approximately the above properties, were isolated from gelatin by alcohol precipitation at different temperatures. 5. Increasing the temperature and adding neutral salts greatly increase the pressure of the insoluble fraction and have little effect on that of the soluble fraction. 6. Adding increasing amounts of the soluble fraction to the insoluble one results in greater and greater swelling. 7. These results are considered as evidence for the idea that the swelling of gelatin in water or salt solutions is an osmotic phenomenon, and that gelatin consists of a network of an insoluble substance enclosing a solution of a soluble constituent.     

Viscosity of concentrated solutions and of human erythrocyte cytoplasm determined from NMR measurement of molecular correlation times. The dependence of viscosity on cell volume

Metabolically active human erythrocytes were incubated with [alpha-13C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13C-NMR in which the longitudinal relaxation times (T1) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T1 values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T1 estimates the rotational correlation time (tau r) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical ellipsoidal rotor; for glycine the differences in estimates of tau r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T1 measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a 'shape' factor and a 'volume' factor; it was fitted to data relating to the concentration dependence of viscosity measured by both methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the bulk viscosity, it was possible to calculate the NMR 'micro' viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of the probe and solute molecules and must be separately determined for each probe-solute-solvent system. Under various conditions of extracellular osmotic pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T1 of [alpha-13C]glycine. Conversely, we showed that alterations in T1, when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells.     

MOLECULAR WEIGHT,MOLECULAR VOLUME,AND HYDRATION OF PROTEINS IN SOLUTION

1. The gram molecular weight of a substance may be calculated from the osmotic pressure of its solution. 2. The radius of the hydrated molecule and, hence, the gram molecular volume of the hydrated solute may be determined from diffusion measurements. The hydration of the molecules may, therefore, be calculated from osmotic pressure and diffusion measurements. 3. Hydration may also be determined by viscosity measurements. Hydration of crystalline hemoglobin, crystalline trypsin, and gelatin have been determined by these methods and found to be as follows: See PDF for Structure     

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van''t Hoff''s law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H₂O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin.     

AMPHOTERIC COLLOIDS : II. VOLUMETRIC ANALYSIS OF ION-PROTEIN COMPOUNDS; THE SIGNIFICANCE OF THE ISOELECTRIC POINT FOR THE PURIFICATION OF AMPHOTERIC COLLOIDS.

1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.     

Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. II. Electrostatic interactions in individual fatty acid binding sites

13C NMR chemical shift results as a function of pH for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented. For octanoic acid bound to BSA (6:1, mol/mol), the chemical shift of the only FA carboxyl resonance (designated as peak c), plotted as a function of pH, exhibited a complete sigmoidal titration curve that deviated in shape from a corresponding theoretical Henderson-Hasselbach curve. However, FA carboxyl chemical shift plotted as a function of added HCl yielded a linear titration curve analogous to those obtained for protein-free monomeric fatty acid (FA) in water. The apparent pK of BSA-bound octanoic acid was 4.3 +/- 0.2. However, the intrinsic pK (corrected for electrostatic effects resulting from the net positive charge on BSA) was approximately 4.8, a value identical to that obtained for monomeric octanoic acid in water in the absence of protein. For long-chain FA (greater than or equal to 12 carbons) bound to BSA (6:1, mol/mol), chemical shift titration curves for peak c were similar to those obtained for octanoic acid/BSA. However, the four additional FA carboxyl resonances observed (designated as peaks a, b, b', and d) exhibited no change in chemical shift between pH 8 and 3. For C14.0 X BSA complexes (3:1 and 6:1, mol/mol) peaks b' and a exhibited chemical shift changes between pH 8.8 and 11.5 concomitant with chemical shift changes in the epsilon-carbon (lysine) resonance. In contrast, peaks c and d exhibited no change and peak b only a slight change in chemical shift over the same pH range. We conclude: the carboxyl groups of bound FA represented by peaks a, b, b', and d were involved in ion pair electrostatic interactions with positively charged amino acyl residues on BSA; the carboxyl groups of bound FA represented by peak c were not involved in electrostatic interactions with BSA; the similarity of the titration curves of peak c for BSA-bound octanoic acid and long-chain FA suggested that short-chain and long-chain FA represented by peak c were bound to the same binding site(s) on BSA; bound FA represented by peaks b' and a (but not d or b) were directly adjacent to BSA lysine residues. We present a model which correlates NMR peaks b, b', and d with the putative locations of three individual high-affinity binding sites in a three-dimensional model of BSA.     

THE SWELLING AND OSMOTIC PRESSURE OF GELATIN IN SALT SOLUTIONS

1. The swelling and the osmotic pressure of gelatin at pH 4.7 have been measured in the presence of a number of salts. 2. The effect of the salts on the swelling is closely paralleled by the effect on the osmotic pressure, and the bulk modulus of the gelatin particles calculated from these figures is constant up to an increase in volume of about 800 per cent. As soon as any of the salts increase the swelling beyond this point, the bulk. modulus decreases. This is interpreted as showing that the elastic limit has been exceeded. 3. Gelatin swollen in acid returns to its original volume after removal of the acid, while gelatin swollen in salt solution does not do so. This is the expected result if, as stated above, the elastic limit had been exceeded in the salt solution. 4. The modulus of elasticity of gelatin swollen in salt solutions varies in the same way as the bulk modulus calculated from the osmotic pressure and the swelling. 5. The increase in osmotic pressure caused by the salt is reversible on removal of the salt. 6. The observed osmotic pressure is much greater than the osmotic pressure calculated from the Donnan equilibrium except in the case of AlCl₃, where the calculated and observed pressures agree quite closely. 7. The increase in swelling in salt solutions is due to an increase in osmotic pressure. This increase is probably due to a change in the osmotic pressure of the gelatin itself rather than to a difference in ion concentration.     

Extraction and electrospinning of gelatin from fish skin

Ultra-fine gelatin fibers were successfully fabricated by electrospinning from the solutions of Nile tilapia (Oreochromis niloticus) skin-extracted gelatin in either acetic acid or formic acid aqueous solutions. The extracted gelatin contained 7.3% moisture, 89.4% protein, 0.3% lipid, and 0.4% ash contents (on the basis of wet weight), while the bloom gel strength, the shear viscosity, and the pH values were 328 g, 17.8 mPa s, and 5.0, respectively. Both the acid concentration and the concentration of the gelatin solutions strongly influenced the properties of the as-prepared solutions and the obtained gelatin fibers. At low acid concentrations (i.e., 15% (w/v) extracted gelatin solutions in 10 and 20% (v/v) acetic acid solvents or 10-60% (v/v) formic acid solvents), a combination between smooth and beaded fibers was observed. At low concentrations of the gelatin solutions in either 40% (v/v) acetic acid solvent or 80% (v/v) formic acid solvent (i.e., 5-11%, w/v), either discrete beads or beaded fibers were obtained, while, at higher concentrations (i.e., 14-29%, w/v), only smooth or a combination of smooth and beaded fibers were obtained. The average diameters of the obtained fibers, regardless of the types of the acid solvents used, ranged between 109 and 761 nm. Lastly, cross-linking of the obtained gelatin fiber mats with glutaraldehyde vapor caused slight shrinkage from their original dimension, and the cross-linked gelatin fiber mats became stiffer.     

ELECTROKINETIC PHENOMENA : X. ELECTRIC MOBILITY AND CHARGE OF PROTEINS IN ALCOHOL-WATER MIXTURES

1. The electrophoretic velocities of gelatin-, egg-albumin-, and gliadin-covered quartz particles in various alcohol-water solutions are, within the limits employed in usual experimental procedures, proportional to the field strength. 2. The electrophoretic mobilities of small, irregularly shaped quartz particles covered with an adsorbed film of protein in alcohol-water solutions are equal to the electroosmotic mobilities of the liquid past similarly coated flat surfaces. Hence the size and shape of such particles does not influence their mobilities, which depend entirely on the protein film. 3. The corrected mobility and hence presumably the charge of gelatin-covered quartz particles in solutions containing 35 per cent ethyl alcohol is proportional to the combining power of the gelatin; therefore the gelatin is adsorbed with the active groups oriented toward the liquid. The same is true in 60 per cent alcohol. 4. The charge calculated by means of the Debye-Henry approximation from the mobility of gelatin in solutions containing up to 35 per cent ethyl alcohol is, in the neighborhood of the isoelectric point, proportional to the combining power of the gelatin. Therefore the dielectric constant and the viscosity of the bulk of the medium may be used in the Debye-Henry approximation Q = 6 π η r v_m (1 + κ r) to predict changes in charge from mobility. 5. In the neighborhood of the isoelectric point gelatin is probably completely ionized in buffered ethyl alcohol-water mixtures up to 60 per cent alcohol. 6. In the presence of ethyl alcohol the isoelectric point of gelatin is shifted toward smaller hydrogen ion activities. This shift, like that caused by alcohol in the isoelectric points of certain amino acids, is approximately linearly related to the dielectric constant of the medium.     

Intrinsic viscosities and other solution properties of sugars and their possible relation to sweetness

The intrinsic viscosities, apparent molar volumes, apparentspecific volumes and apparent solution parachors of selectedsugars were determined with the aim of investigating the possiblerelationship of these solution properties to the published sweetnessvalues of sugars. For a given sugar, there is no clear relationshipbetween its intrinsic viscosity and other solution propertiesstudied. The values of apparent specific volume and intrinsicviscosity differ greatly even though both parameters reflectthe amount of water surrounding 1 g of the solute. There isalso no observable correlation between the intrinsic viscositiesand the literature values on the relative sweetness of sugars.The intrinsic viscosity of a solute molecule reflects its extentof hydration. It is greatly influenced by the conformation andconfiguration of the sugar molecules. Equatorial OH groups aremore favourable for hydration than the axial OH groups. Thus,the more OH groups in the equatorial position, the higher theintrinsic viscosity.     

Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation

The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation.The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS³ concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol.Gelation is not accompanied by a detectable volume change (limits 5 × 10^?5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm³/mol when the aggregates represent one half of the HbS available for polymerization.     

The mechanism of ion selectivity of OmpF-porin pores of Escherichia coli

The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis. (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.     

Determinants of epithelial cell volume

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.